The publication and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference.
Human osteocalcin (hOC), also designated bone Gla protein (BGP), is the most abundant noncollagenous protein synthesized by bone osteoblast (Poser et. al. J Biol Chem 1980; 255: 8685–91). Although most of the synthesized osteocalcin is absorbed to bone hydroxyapatite by γ-carboxylated glutamic acids (Gla), a small part of it leaks into the blood stream where it can be detected (Price et al. J Biol Chem 1981; 256: 12760–6). Part of the hOC found in blood is also thought to originate from the resorption process, when the hOC inside the bone tissues is released during bone degradation (Gundberg and Weinstein J Clin Invest 1986; 77: 1762–7). Levels of circulating hOC have been widely used in the clinical investigations as a marker of bone formation (Power and Fottrell Crit Rev Clin Lab Sci 1991; 28: 287–335) and serum hOC levels have been shown to correlate with bone mineral density measurements in many situations (Yasamura et al. J Clin Endocrinol Metab 1987; 64: 681–5).
The discordant results obtained from different hOC assays have hindered widespread usage of hOC in clinical applications (Masters et al. Clin Chem 1994; 40: 358–63, Deftos et al. Clin Chem 1992; 38: 2318–21, Delmas et al. J Bone Miner Res 1990; 5: 5–11 and Diego et al. 1994; 40: 2071–7). This phenomenon could partly be explained due to different assay formats i.e. sandwich vs. competitive assays or due to different detection techniques. Presently no calibration standard is available. However, even if the same standard preparation is used, hOC levels measured in different laboratories cannot be directly compared (Delmas et al. J Bone Miner Res 1990). The diversity of hOC molecule itself in circulation has an evident contribution to its immunoreactivity in various assays. The vitamin K dependent γ-carboxylation degree of the glutamic acid residue varies (Poser et. al. J Biol Chem 1980; 255: 8685–91). Impairment γ-carboxylation of hOC purified from bone has been indicated by Cairns and Price, J Bone Min Res 1994; 9: 1989–97 and confirmed in our studies (Hellman et al. J Bone Miner Res 1996; 11: 1165–75). When Ca2+ binds to Gla residues an α-helix structure is known to form (Hauschka and Carr, Biochemistry 1982; 21: 2538–47 and Atkinson et al. Eur J Biochem 1995; 232: 515–21). Upon removal of Ca2+ with EDTA this helical conformation is destroyed. The conformation of decarboxylated OC lies somewhere between the random coil and helical form. Thus, in solution the peptide occurs as a flexible structure and a single conformation cannot be defined for it (Atkinson et al. Eur J Biochem 1995; 232: 515–21). Peptide bonds between arginine residues 19 and 20 and between residues 43 and 44 are susceptible to tryptic hydrolysis leading to peptides 1–19, 20–43, 45–49, 1–43, and 20–49 which may be the main products of hOC breakdown in the circulation (Farrugia and Melick, Calcif Tissue Int 1986; 39: 234–8, Hellman et al. J Bone Miner Res 1996; 11: 1165–75 and Garnero et al. J Bone Miner Res 1994; 9: 255–4). Multiple immunoreactive forms of hOC have been discovered in circulation (Garnero et al. J Bone Miner Res 1994; 9: 255–4) and also in urine (Matikainen et al. J Bone Miner Res 1999; 14: 431–8, Taylor et al. J Clin Endocrin Metab 1990; 70: 467–72). The fragments of hOC can be produced either during osteoclastic degradation of bone matrix or as the result of the catabolic breakdown of the circulating protein after synthesis by osteoblasts.
The degree of vitamin K-dependent γ-carboxylation in the glutamic acid residues varies, so the circulating hOC is a pool of hOC molecules with variable γ-carboxylation (Cairns and Price J Bone Min Res 1994; 9: 1989–97). During the recent years the relevance of the γ-carboxylation degree of osteocalcin for predicting the bone turnover status has been investigated. Osteocalcin is known to be attached to the bone mineral content, hydroxyapatite, by γ-carboxylated glutamic acids. Hydroxyapatite has thus been used to distinguish the fully γ-carboxylated form of protein from non- or undercarboxylated (uchOC) forms (Price et al. J Biol Chem 1981; 256: 12760–6). The hydroxyapatite binding capacity of the circulating osteocalcin has been reported to be abnormally low in elderly people when compared to the premenopausal group (Knapen et al. Ann Intern Med 1989; 111: 1001–5, Merle and Delmas Bone Miner 1990; 11: 237–45). Szulc et al. (J Clin Invest 1993; 91: 1769–74, J Bone Miner Res 1994; 9: 1591–5, Bone 1996; 18: 487–8) have shown that circulating uchOC is a marker of the risk of hip fracture in a population of institutionalized women and that the BMD (bone mineral density) is significantly decreased in women with elevated uchOC. However, the hydroxyapatite-based assays provide only a crude estimation of the degree of carboxylation. Complete separation of the two forms is difficult due to incomplete binding of carboxylated forms at lower concentrations and particularly because of nonspecific binding of undercarboxylated osteocalcin at higher concentrations (Merle and Delmas Bone Miner 1990; 11: 237–45, Käkönen et al. Protein Expres Purif 1996; 8: 137–44, Szulc et al. J Clin Invest 1993; 91: 1769–74). Moreover, the methods could be sensitive to changes in buffer conditions and quality of the hydroxyapatite reagents employed. Gundberg et al. showed that N-terminal binds lower. Recently, Vergnaud et al. (J Clin Endocrinol Metab 1997; 82: 719–24) developed a new ELISA for uchOC based on Mabs, with low cross-reactivity to carboxylated hOC. Using this specific immunoassay they showed that uchOC, but not total hOC, predicted hip fracture risk independently of bone mass in elderly women drawn from the general population. Osteoporotic fractures have serious consequences among the elderly. They result in high mortality, frequent hospitalization, high health care costs, functional impairment, pain and reduced quality of life (Ross Arch Intern Med 1996; 156: 1399–411). Therefore, easily implemented valid methods to assess the risk of fracture are needed.
Low bone density predicts the occurrence of fracture (Hui et al. Ann Intern Med 1989; 111: 355–61, Melton et al. J Bone Miner Res 1993; 8: 1227–33) and hip fracture (Cummings et al. Lancet 1993; 341: 72–5). Serum osteocalcin as a measure of increased bone turnover is a determinant of osteoporosis in postmenopausal women, and the serum concentrations of osteocalcin increase with advancing age (Garnero et al. J Bone Miner Res 1996; 11: 337–49, Melton et al. J Bone Miner Res 1997; 12: 1083–91), in both men and women (Epstein et al. Lancet 1984; 307–10) Results concerning the relationship between osteocalcin and the occurrence of fracture are contradictory, as some authors suggest that there is a positive association (Riis et al. Bone 1996; 19: 9–12), while others suggest that there is no association (Åkesson et al. J Bone Miner Res 1995; 10: 1823–9, Garnero et al. J Bone Miner Res 1996; 11: 1531–8).
The degree of the vitamin K-dependent γ-carboxylation of osteocalcin (Plantalech et al. J Bone Miner Res 1991; 6: 1211–6, Obrant et al., J Bone Miner Res 1999; 14: 555–60) becomes reduced with aging (Knapen et al. Ann Intern Med 1989; 111: 1001–5, Merle and Delmas Bone Min 1990; 11: 237–45) and by affecting the calcium-binding capacity of the protein may lead to osteopenia (Pastoreau et al. J Bone Miner Res 1993; 8: 1417–26). In line with these findings, high proportions of under-carboxylated osteocalcin are associated with the occurrence of hip fractures in the institutionalized elderly (Szulc et al. J Clin Invest 1993; 91: 1769–74). This finding was supported by the results of a case-control study within a large prospective study among healthy elderly community-living women (Vergnaud et al. J Clin Endocrinol Metab 1997; 82: 719–24).